Identification and characterization of reflectin proteins from squid reflective tissues

ABSTRACT

A family of reflectin proteins is identified herein that is deposited in flat, structural platelets in reflective tissues of the squid  Euprymna scolopes . These proteins are encoded by at least six genes in three subfamilies and have no reported homologues outside of squids. Reflectins possess 5 repeating domains, that are remarkably conserved among members of the family. The proteins have a highly unusual composition with four relatively rare residues (tyrosine, methionine, arginine, and tryptophan) comprising ˜57% of a reflectin, and several common residues (alanine, isoleucine, leucine, and lysine) occurring in none of the family members. These protein-based reflectors in squids provide a striking example of nanofabrication in animal systems.

PRIORITY

This application claims priority to U.S. Provisional Application60/549,733, filed Mar. 2, 2004, herein incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made in part with Government support under Grant No.IBN 0211673, and AI50661 awarded by the National Institutes of Health.The Government may have certain rights in this invention.

FIELD OF THE INVENTION

The invention relates generally to a new family of proteins that composea subcellular structure that confers reflectivity to squid tissues. Morespecifically, the invention relates to squid reflectin proteins andactive portions and repeat units thereof.

BACKGROUND OF THE INVENTION

The biological world is an arena of nanofabrication, one that can betapped for information about constraints on the design and production ofsmall-scale materials. Among the most intricate of natural nanoscalematerials are those that modulate light, such as the lenses, irises, andreflectors of animals (Vukusic, et al. 2003 Nature 424, p. 852).Reflective tissues are prevalent across the animal kingdom, beingparticularly conspicuous in species that live in the visuallyhomogeneous pelagic environments of the ocean. In these habitats,reflectors often function in camouflaging by modulating incidentsunlight or bioluminescence (Johnsen, et al. Proc. Royal Soc. London. B2001, 269, p. 243; Johnsen, et al. Limnol. Oceanogr. 2003, 48, p. 1277).Reflectivity in animal tissues is achieved by the deposition of flat,insoluble, structural platelets of high refractive index that alternatein layers with materials of low refractive index. This arrangementcreates thin-film interference, that results in reflection of some orall of the incident light (Land, et al. Prog. Biophys. Molec. Biol.1972, 24, p. 75). In aquatic animals, reflector platelets are most oftencomposed of purine crystals, particularly guanine and hypoxanthine(Denton, et al. Proc. Roy. Soc. Lond. A. 1971, 178, 43). In contrast,cephalopod reflector platelets do not contain these purines and studiesof their biochemical and biophysical characteristics have suggested thatthey are composed of protein (Cooper, et al. Cell Tissue Res. 1990, 259,p. 15). However, the composition of cephalopod reflector platelets hasnever been definitively characterized (Cloney, et al. Amer. Zool., 1983,23, p. 581). Each of the cited references herein are incorporated byreference in its entirety.

SUMMARY OF THE INVENTION

One embodiment provides for an isolated reflectin polypeptide having asequence with at least about 75% identity to SEQ ID NO:2, including 85%identity to SEQ ID NO:2. In some embodiments, the polypeptide has apredicted isoelectric point above 8.0.

Other embodiments provide for an isolated polynucleotide encoding areflectin polypeptide, the polynucleotide having a sequence with atleast about 65% identity to SEQ ID NO:1, including 77% identity and 85%identity.

Other embodiments provide for an isolated polypeptide having and leastone and no more than four repeats of an amino acid sequence having themotif [α(X)_(4/5)MD(X)₅MD(X)_(3/4)], wherein α is MD, FD, or null; Xrepresents any amino acid; the subscripted numbers represent the numberof amino acids at that position; and the slash represents “or.” In someembodiments the amino acid sequence is selected from the groupconsisting of: SEQ ID NOs: 15-30 and any combination thereof. In otherembodiments the isolated polpeptide has an activity of a reflectinprotein.

Other embodiments provide for an isolated polypeptide having six or morerepeats of an amino acid sequence having the motif[α(X)_(4/5)MD(X)₅MD(X)_(3/4)], wherein α is MD, FD, or null; Xrepresents any amino acid; the subscripted numbers represent the numberof amino acids at that position; and the slash represents “or.” In someembodiments the amino acid sequence is selected from the groupconsisting of: SEQ ID NOs:15-30, and any combination thereof.

Other embodiments provide for a biomimetic reflective material having afirst component, the first component having at least one polypeptideselected from (a) a reflectin polypeptide; (b) a polypeptide having atleast one and not more than four repeat units of a reflectinpolypeptide; (c) a polypeptide having at least six repeat units of areflectin polypeptide; (d) an active or functional homologue orrecombinant form of any of (a) through (c); and (e) any combination of(a) through (d); the first component being in combination with at leasta second component compatible with the first component, such that thecombination forms a biomimetic reflective material. In some embodiments,the biomimetic reflective material includes a metal ion. In otherembodiments, the material has at least a first and a second refractivestate, wherein the material in the first refractive state has arefractive index that is different from a refractive index of thematerial in the second state.

Other embodiments provide for a method of producing a biomimeticreflective material, by providing a first component having at least onepolypeptide (a)-(e) where (a) a reflectin polypeptide; (b) a polypeptidehaving at least one and not more than four repeat units of a reflectinpolypeptide; (c) a polypeptide having at least six repeat units of areflectin polypeptide; (d) an active or functional homologue orrecombinant form of any of (a) through (c); and (e) any combination of(a) through (d); combining the first component with at least a secondcomponent to form a biomimetic reflective material. In some embodiments,the second component is a metal, an ion, a polymer, a fabric, a crystal,a fiber, a plastic or any other suitable material.

Further embodiments include a method of producing a biomimeticreflective material, by causing expression in a cell, of at least onepolypeptide selected from: (a) a reflectin polypeptide; (b) apolypeptide having at least one and not more than four repeat units of areflectin polypeptide; (c) a polypeptide comprising at least six repeatunits of a reflectin polypeptide; (d) an active or functional homologueor recombinant form of any of (a) through (c); and (e) any combinationof (a) through (d); using the cell or a fragment or extract thereof inproducing a biomimetic reflective material. In some embodiments, thecell is a plant cell, a bacterial cell; a fungal cell, or an animalcell.

Further embodiments provide for a method of modifying a refractive indexof a reflectin, by: providing a reflectin polypeptide in a compositioncompatible with a metal ion, wherein the composition has a firstrefractive index in absence of the metal ion; adding the metal ion tothe composition, wherein the composition has a second refractive indexin presence of the metal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Reflective tissues in Euprymna scolopes. (A) The locations ofreflective (dg, digestive gland; er, eye reflector; lor, light organreflector; m, mantle) and non-reflective (el, eye lens; g, gill) tissuesof E. scolopes are revealed by a ventral dissection of an adult animal;inset, an adult animal. (B) A light micrograph of a cross section(location at orange line in panel A) of the light organ. The centralepithelium (e) is surrounded by the reflector (lor), that is in turnsurrounded by ink sac diverticula (is). Lens tissue (lol) is located onthe ventral surface of the light organ. (C) Transmission electronmicrograph (TEM) of the boxed area in B. Stacks of electron-densereflector platelets (p) abut connective tissue (ct) and the ink sac withits secreted ink granules (ig). (D) Higher magnification TEM of thelight organ reflector (LOR) platelets. (E) Silver-stained SDS-PAGE gelof protein extracts from LORs. LOR tissue extracts were prepared asdescribed in the Examples. lane 1, molecular mass markers (std)expressed in kDa; lane 2, total homogenate of LOR (total); lane 3,supernatant fraction of LOR extracted in PBS (soluble); lane 4,supernatant of pellet from PBS-extracted LOR re-extracted with 2% SDS(pellet); lane 5, purified reflectins (purified). 5 μg of protein loadedin lanes 2-4 samples; 3 μg loaded in lane 5. (E′) Higher magnificationof reflectin bands, showing the presence of 3 protein species.

FIG. 2. Localization of reflectins in E. scolopes. (A)Immunocytochemistry at the light level. One-μm sections of wholejuvenile squid mounted on gelatin-coated glass slides were incubated ineither preimmune serum (preimmune) or reflectin antiserum(anti-reflectin). Fifteen-nm gold beads conjugated to goat anti-rabbitIgGs were used as secondary antibodies and sections were silver enhanced(Silver Enhancer Kit; Sigma-Aldrich) to allow detection of the goldbeads by light microscopy. The sections were then either counterstainedwith 1% acid fuchsin (+cs) or left unstained (−cs) and viewed bydifferential interference microscopy. is, ink sac; lor, reflector; e,central epithelial tissue. (A′) Immunocytochemistry at the TEM level.Ultrathin sections of whole juvenile squid mounted on nickel grids wereincubated in either preimmune serum or reflectin antiserum. Fifteen-nmgold beads conjugated to goat anti-rabbit IgGs were used as secondaryantibodies. Inset, higher magnification showing the labeling of anindividual platelet. p, platelets; ct, connective tissue; ig, inkgranules. (B) Silver-stained SDS-PAGE (lanes 1-9) and immunoblotanalyses (lanes 10-18) of 2% SDS-extracted proteins from pellets ofaqueous-buffer extractions of reflective and non-reflective squidtissues; 2.5 μg total protein loaded per lane. std, molecular massstandards in kDa; arrowhead indicates the position on the gels wherereflectins resolve. dig. gland, digestive gland; l.o. lens, light organlens; l.o. reflector, light organ reflector.

FIG. 3. Reflectins are composed of repeating domains, as predicted byRADAR (Rapid Detection and Alignment of Repeats). (A) Upper panel, theentire amino acid sequence of reflectin la with repeat regions indicatedin grey and amino acids excluded from the repeats in black (SEQ IDNO:2). Numbers and arrows indicate the number and direction of therepeats. Lower panel, the RADAR output. The fourth repeat was used asthe template repeat. Score, “score of each repeat unit when scoredagainst the whole repeat” (RADAR); Std Dev, the number of standarddeviations above the mean for shuffled sequences scored against the sameprofile. (B) Schematic showing the positioning of the repeats (grayboxes) and the conserved subdomains (hatched boxes; SD1-SD5) from arepresentative reflectin. The subdomains are also outlined in the RADARalignment in panel 3A (SEQ ID NOs:16-24). The subdomain amino acidalignments among all reflecting are shown under each subdomain. SD1=SEQID NO:16, SD2=SEQ ID NO:17, SD3=SEQ ID NO:18 and 19, SD4=SEQ ID NO:20and 21, and SD5=SEQ ID NOs: 22, 23, and 24.

FIG. 4. Amino acid alignments, comparisons, and compositions of thederived amino acid sequences for E. scolopes reflecting and L. forbesimrrp1 (Lf). (A) Alignment (Clustal V) of reflectin proteins 1a-3a withL. forbesi mrrp1. 1a is SEQ ID NO:2, 1b is SEQ ID NO:4, 2a is SEQ IDNO:6, 2b is SEQ ID NO:8, 2c is SEQ ID NO:10, 3a is SEQ ID NO:12, and Lfis SEQ ID NO:14. The black bars are located above the tryptic peptidesthat were sequenced (SEQ ID NOs: 31-33); *, residues are identical inall sequences in the alignment; colon (:), are conserved substitutionspresent in the alignment; period (.), are semi-conserved substitutionspresent in the alignment. (B) Pairwise comparison of the reflectins andL. forbesi mrrp1 expressed as percent identity. (C) Amino acidcomposition of representatives of the E. scolopes reflectin family. # isthe number of times each amino acid occurs in each protein; % is thepercent of each amino acid out of the total number of amino acids thatoccur in each protein.

FIGS. 5 a and 5 b. FIG. 5 a is a ClustalW alignment of E. scolopesreflectins 1a, 2a and 3a and L. forbesi mrrp1 nucleotide sequences (SEQID NOs: 1, 5, 11 and 13). These sequences share 62.5% identity. FIG. 5 bis a ClustalW alignment of E. scolopes reflectins 1a, 1b, 2a, 2b, 2c,2d, and 3a nucleotide sequences (SEQ ID NOs: 1, 3, 5, 7, 45 and 9).

FIG. 6. Ribbon structure representing the reflectin 1A gene from E.scolopes. The Reflectin Repeat Peptide (RRP) amino acid sequence usedherein is shown below the ribbon structure (SEQ ID NO:15).

FIG. 7. (Left) Circular Dichroism of the RRP showing both near andfar-UV absorbance spectra. (Right) X-ray diffraction of RRP dried in aglass capillary. The diffraction pattern is representative of beta-sheetsecondary structure.

FIG. 8. (Left) LVTEM image of RRP that had recently been resuspended inwater. (Middle) LVTEM image of the same RRP after it was allowed to agefor approximately 6 weeks and then spotted on the TEM grid. (Right) AFMtopography image and FFT of freshly resuspended RRP.

FIG. 9. (Left and Middle) HVTEM image of aged RRP showing spherical andfibril substructures at different magnifications. (Right) Electrondiffraction pattern from the sample on the right. A diffraction aperturewas used to limit the field as to only include the proteinaceousmaterial.

FIG. 10. (Left) LVTEM of RRP mixed with ZnSO₄. (Middle and Right) HVTEMof RRP mixed AuCl₄ at two different magnifications.

FIG. 11. (A) Optical images of precipitated RRP from crystal trials attwo different levels of illumination. Similar precipitation occurred in20% of conditions tested. Images are 3 cm×3 cm. (B) Optical imagesrotated 30° using a cross-polarized light microscope showingbirefringence of the precipitated RRP from A.

FIG. 12. Recombinant expression of the reflectin protein with (right)and without (left) the N-terminally fused HIS tag. Strong induction ofthe appropriately sized band on the SDS-page gel can be seen with andwithout the affinity tag.

FIG. 13 is the nucleotide sequence of the Reflectin la sequence usingE.coli codon usage (SEQ ID NO:44) to produce the polypeptide sequence ofReflectin 1a (SEQ ID NO:2).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A family of reflectin proteins is described herein; the proteins aredeposited in flat, structural platelets in reflective tissues of thesquid Euprymna scolopes. These proteins are encoded by at least sixgenes in three subfamilies and have no reported homologues outside ofsquids. Reflectins possess 5 repeating domains that are remarkablyconserved among members of the family. The proteins have a highlyunusual composition with four relatively rare residues (tyrosine,methionine, arginine, and tryptophan) comprising ˜57% of a reflectin,and several common residues (alanine, isoleucine, leucine, and lysine)occurring in none of the family members. These protein-based reflectorsin squids provide a striking example of nanofabrication in animalsystems.

Identification of the reflectin proteins associated with reflectivetissues of E. scolopes has led to data on amino acid composition andsequence that are unique to electron dense reflective tissues of thisspecies. Analysis of these sequences has shown the composition of theprotein to contain a high percentage of arginine, tyrosine, methionine,and tryptophan residues. Within each of the identified reflectinproteins there exists five repeating domains that show strong sequenceconservation. Repeat domains have been the hallmark of many structuralproteins identified throughout nature, and typically represent thecatalytic, or functional, element of the protein. In an effort toelucidate the nature of the reflectin repeat peptides (RRP), the RRPfrom the third repeat region from the reflectin 1a protein (FIG. 6) wasstudied, having the sequence MDMSNYMDMYGRYMDRWG (SEQ ID NO:15). The datashow that the RRP has reflective activity and also show the secondarystructure and properties of the protein in solution and in the presenceof metal ions. The data suggest that the RRP can be used in lieu of thewhole protein for the same uses.

In vivo, it is believed that the reflecting platelets function by actingas Bragg reflectors with alternating regions of high and low index ofrefraction materials. The generation of a proteinaceous matrix with ahigh refractive index is dependent on a number of variables and includesamino acid composition, concentration and crystallinity of the material,and addition of materials such as inorganic metals or in vivo associatedligands/proteins that can complex with the reflectin proteins. Inaddition, there may exist molecular level organization of the reflectinprotein to optimize the overall effective refractive index. Thesevariables are explored through structural and optical characterizationof the RRP. Investigation into the discovery of a protein-basedreflective material represents a paradigm shift in how structuralcoloration is viewed. Both the overall bulk materials and microstructurecontribute to the reflective ability of these structures, and thesemechanisms work cooperatively. While static reflection characterized inthis work differs mechanistically from that of dynamic iridophoretissues, the latter most likely derives its function through molecularmanipulation of a similar bulk material described herein. The ability torearrange substructures, alter binding of inorganics and associatedproteins, and/or control crystallinity of the bulk can represent someways in which dynamic reflection can be controlled. It is likely thatthe overarching principles and structure in both dynamic and staticsystems are related and should be represented by a conservation of aminoacid sequences of the proteins from different species (see also Crookes,et al. 2004 Science Vol. 303, page 235, incorporated by reference in itsentirety).

Reflective Tissues

The Hawaiian bobtail squid Euprymna scolopes (Cephalopoda:Sepiolidae;FIG. 1A) is similar to other cephalopod species that have been studiedin having both variably reflective tissues, such as the skin of themantle, and statically reflective tissues, such as those associated withthe eye, digestive gland, and light organ. The reflector of the bibbedlight organ is a particularly well developed tissue (FIG. 1A-D) thatmodulates the luminescence produced by a population of the symbioticbacterium Vibrio fischeri. On each side of the adult light organ,symbiont-containing epithelial tissue comprises a core that issurrounded by the thick silvery reflector. Together with amuscle-derived lens, these dioptrics function to direct the bacterialluminescence ventrally. Consistent with reflectors in other animals, thelight organ reflector (LOR) tissue is composed of a thick layer ofplatelets (FIG. 1C-D).

The Reflectin Proteins

The seven novel reflectin proteins from squid, and their nucleotidesequences are disclosed herein (see the Examples). Thus, someembodiments of the present invention include one or more novel reflectinpolypeptides and/or polynucleotides encoding such polypeptides.

In some embodiments, the reflectin polypeptide is at least about 72%identical to at least one of the reflectin proteins from E. scolopes(SEQ ID NOs: 2, 4, 6, 8, 10, 12 and 47), including but not limited toabout 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,and 100%. In further embodiments, the polypeptide is at least about 75%identical to at least one of the reflectin proteins from E. scolopes(SEQ ID NOS: 2, 4, 6, 8, 10, 12 and 47). In further embodiments, thepolypeptide is a functional reflectin. In some embodiments, byfunctional is meant that a polypeptide has the function or activity of areflectin. In further embodiments, a functional polypeptide shows agolden yellow color upon SDS-PAGE electrophoresis and silver staining.In further embodiments, a functional polypeptide acts as a BRAGGreflector. In further embodiments, a functional polypeptide has a highrefractive index. In further embodiments, a functional polypeptide cancomplex with inorganic metals. In further embodiments a functionalpolypeptide is active when it has at least one of the above activitiesand/or qualities.

Further embodiments are polynucleotides that encode the polypeptiderecited above. In some embodiments, the polynucleotide is a naturalsequence from a squid genome. In further embodiments, the polynucleotideis a derived sequence in which the codon usage for E. coli or analternative organism is used to express a polypeptide that is at least72.5% identical to any of SEQ ID NOS:2, 4, 6, 8, 10, 12 and 47. Infurther embodiments, the polynucleotide is at least about 65% identicalto the polynucleotide sequence from E. scolopes reflectin proteins 1a,1b, 2a, 2b, 2c and 3a (SEQ ID NOS: 1, 3, 5, 7, 9, 11 and 46). In furtherembodiments, the polynucleotide is at least about 70% identical,including but not limited to: 75%, 77%, 80%, 85%, 90%, 95%, 97.5%, and99%. In further embodiments, the polynucleotide sequence encodes anactive or functional reflectin protein as described above.

As described herein, each reflectin protein is composed of a series offive repeats (designated RRPs). Thus, further embodiments arepolypeptides or corresponding polynucleotide sequences that areengineered or truncated in such a way as to remove one or more of theRRPs. Further embodiments are polypeptides or correspondingpolynucleotide sequences that have one or more of the RRP sequencesremoved internally. This can result in an active or functionalpolypeptide that simply has fewer repeat units but still retainsfunction.

Reflective Repeat Peptide (RRP)

In some embodiments, the RRP is any amino acid sequence corresponding tothe motif [α(X)_(4/5)MD(X)₅MD(X)_(3/4)], wherein α is MD, FD, or null; Xrepresents any amino acid; the subscripted numbers represent the numberof amino acids at that position and the slash represents “or.” Forexample (X)_(4/5) means that either four or five amino acids can be atthat position, and those four or five amino acids can be any amino acidsin any order or combination. Various permutations of the single repeatpeptide include SEQ ID NOs: 15-30.

A highly conserved sequence was identified within the RRP motif as shownin Table 1. Thus, in some embodiments, the RRP sequence comprises themotif [MDMQGRY/W]. In further embodiments, the RRP comprises any aminoacid sequence corresponding to the motif [MDMQGRY/W] or any variantswith conserved amino acids within that sequence, wherein the last aminoacid is either Y or W. The sequence may include other amino acids at theN- or C-terminus, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, and 20 additional residues. In someembodiments, the amino acids at the N- and/or C-terminus are selectedfrom those in SEQ ID NOs: 15-30. TABLE 1 M S R M T M D F Q G R Y M D S QG R M D M S G Y Q M D M S G R W M D M Q G R M D M S N Y S M D M Y G R YM D R W G R M D M S G Y Q M D M Q G R Y M D R W G R F D M S N W Q M D MQ G R W M D N Q G R F G M S N W Q M D M Q G R W M D N Q G R M D Y S N YQ M D M Q G R Y M D Q G R M D Y S N W Q M D M Q G R W M D M Q G R M D YS N Y Q M D M Q G R Y M D M Q G R F D M S N W Q M D M Q G R Y M D Q Y GM D M S N Y S M D M Q G R W M D N Q G R M D M S G Y Q M D M Q G R W M DM Q G R M S R M T M D F Q G R Y M D R W G R F D M S N W Q M D M Q G R YM D Q Y G R F D M S R M T M D F Q G R Y M D S Q G R

Because the RRPs are the functional units of the protein, anymodifications to the protein sequence that conserve at least one of theRRP sequences can result in an active or functional polypeptide.Likewise, any modifications to a polynucleotide encoding the protein,when such modifications conserve at least one RRP sequence, results in apolynucleotide encoding an active or functional polypeptide. Thus, afurther embodiment is any mutated form of the polypeptides and/orpolynucleotides that results in at least one conserved RRP within thesequence. Further embodiments include a polypeptide or an encodingpolynucleotide, with a sequence that results in a modified RRP thatmaintains reflective and/or structural characteristics. In someembodiments, permutations and variants include any changes that stillproduce an active reflectin and/or reflectin repeat peptide. In someembodiments, the variants still conform to the formula[α(X)_(4/5)MD(X)₅MD(X)_(3/4)], wherein α is MD, FD, or null; Xrepresents any amino acid; the subscripted numbers represent the numberof amino acids at that position and the slash represents “or.” In afurther embodiment, the variants have substantially the sequences of SEQID NOs: 15-30 with one or more substitutions, insertions or deletionsthat conform to either the formula or one of the RRPs described hereinor a like amino acid at the equivalent position.

A further embodiment is an RRP that comprises the formula[□(X)4/5MD(X)5MD(X)3/4], including but not limited to SEQ ID NOs: 15-30as follows: MSRMTMDFQGRYMDSQGR, (SEQ ID NO:16) MDMSGYQMDMSGRWMDMQGR,(SEQ ID NO:17) MDMSNYSMDMYGRYMDRWGR, (SEQ ID NO:18)MDMSGYQMDMQGRYMDRWGR, (SEQ ID NO:19) FDMSNWQMDMQGRWMDNQGR, (SEQ IDNO:20) FGMSNWQMDMQGRWMDNQGR, (SEQ ID NO:21) MDYSNYQMDMQGRYMDQYG, (SEQ IDNO:22) MDYSNWQMDMQGRWMDMQGR, (SEQ ID NO:23) MDYSNYQMDMQGRYMDMQGR, (SEQID NO:24) FDMSNWQMDMQGRYMDQYG, (SEQ ID NO:25) MDMSNYSMDMQGRWMDNQGR, (SEQID NO:26 MDMSGYQMDMQGRWMDMQGR, (SEQ ID NO:27) MSRMTMDFQGRYMDRWGR, (SEQID NO:28) FDMSNWQMDMQGRYMDQYGR, (SEQ ID NO:29) and FDMSRMTMDFQGRYMDSQGR.(SEQ ID NO:30)

Alternative forms of the RRPs that are still active can be producedusing these known sequences and substituting amino acids at equivalentpositions or producing chimera of the known peptides. For example, thetryptophan at position 6 of SEQ ID NO:23 can be substituted for thetyrosine at position 6 of SEQ ID NO:22. In addition, any amino acidsthat have the same properties can be substituted. Further, an amino acidat position 2 can be added to any RRP peptides as long as the peptidestill conforms to the formula. Other substitutions can be made as longas they conform generally to the formula and still result in an activepolypeptide. In one embodiment, the RRP is MDMSNYMDMYGRYMDRWG (SEQ IDNO:15).

A further embodiment is a polypeptide having, and/or a polynucleotidesequence encoding, one or more of the functional repeat units for thereflectin proteins. In some embodiments, the polypeptide includes 1, 2,3, or 4 RRPs. The RRPs can be any combination or permutation of thoseprovided in SEQ ID NOS: 15-30 and can contain, for example 2 copies ofSEQ ID NO:15, one copy of SEQ ID NO:16 and one copy of SEQ ID NO:17. Insome embodiments, the polypeptide has four copies of SEQ ID NO:15.

In some embodiments, the polynucleotide has one or more copies of anycombination of the functional repeat units as described above. In someembodiments, the polynucleotide encodes a polypeptide having one or morecopies of the functional repeat units. In an alternative embodiment, thepolynucleotide results in one or more separately translated RRPs.

Further embodiments include polypeptides and/or polynucleotides havingor encoding five copies of the RRPs in a combination or arrangement thatis not found in nature. In other words, the repeats are provided in acombination that, while not produced in nature, still results in one ormore active polypeptides.

Further embodiments are polypeptides having, and/or polynucleotidesequences encoding, six or more of the functional repeat units for thereflectin proteins. In some embodiments, the polypeptide includes 6 ormore RRPs, including but not limited to: 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 200, 300, 400, and 500. In further embodiments, thenumber of copies is between about 6 and about 30. In furtherembodiments, the number of copies is more than 6 but less than about1000. The RRPs can be any combination or permutation of those providedin SEQ ID NOS:15-30 and/or functional modifications thereof, and cancontain, for example, 2 copies of SEQ ID NO:15, one copy of SEQ IDNO:16, one copy of SEQ ID NO:17, two copies of SEQ ID NO:18, and onecopy of SEQ ID NO:19, and so on. In some embodiments, the polypeptideincludes six or more copies of SEQ ID NO:15.

In some embodiments, the polynucleotide includes six or more copies ofany combination of the functional repeat units as described above. Insome embodiments, the polynucleotide encodes a polypeptide having six ormore copies of the functional repeat units. In alternative embodiments,the polynucleotide results in six or more separately translated RRPs.

The sequences between the RRPs can be any sequence that does notnegatively affect the secondary or tertiary structure of the RRP and cancontain a promoter region, a stop codon, or an initiation codon. Thus,it is to be understood that the polynucleotide for the RRP protein canbe expressed as a polyprotein containing two or more RRPs or can beexpressed as multiple RRP proteins.

Methods of Expressing Reflectins and/or RRPs

As stated above, the polynucleotide can be expressed as a polyproteincontaining one or more RRPs or can be expressed such that each RRP istranslated separately and/or transcribed separately. Any promoter can beused that will result in expression in the cell of choice.

In some embodiments, the polynucleotide is provided such that the codonusage for the particular cell results in the polypeptide of choice. Forexample, the E. coli codon usage can be used to produce the polypeptideof SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 15-30 or any variant thereof.

Methods of Purifying Reflectins and/or RRPs

In some embodiments, the reflectin protein or RRP unit is purified andstored in a buffer having 0.2% or more SDS or an equivalent detergent,including but not limited to: 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%,1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%,2.2%, 2.3%, 2.4%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5%. In furtherembodiments, the buffer includes SDS at about 0.2 to 2%. In furtherembodiments, the buffer includes nondetergent sulfobetaine (NDSB) 195,201, and/or 256 at a concentration of about 0.1 to about 10 M, includingbut not limited to 9 M, 8 M, 7 M, 6 M, 5 M, 4 M, 3 M, 2 M, 1 M, 0.9 M,0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.4 M, 0.3 M, and 0.2 M. In furtherembodiments the buffer includes CHAPS at a concentration of about 0.1 toabout 10%, including but not limited to: 0.2%, 0.4%, 0.6%, 0.8%, 1%,1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%,6.5%, 7%, 7.5%, 8%, 8.5%, 9%, and 9.5%. In further embodiments, thebuffer contains urea at a concentration of about 3M to about 10M,including but not limited to 4M, 5M, 6M, 7M, 8M, and 9M. In someembodiments the buffer includes DMSO at a concentration of from about60% to about 100%, including but not limited to 70%, 80%, 90%, and 95%.In further embodiments, the buffer includes trifluoroethanol in aconcentration of from about 60% to about 100%, including but not limitedto: 70%, 80%, 85%, 90%, 95%, 97.5%, and 99%. In further embodiments, thebuffer includes EDTA at a concentration of from about 0.2 M to about 3M,including but not limited to 0.3M, 0.4 M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M,1M, 1.5M, 1.75M, 2M, 2.5M, and 2.75M. In further embodiments, the bufferincludes ammonium acetate and/or ammonium sulfate in a concentration ofabout 0.2M to about 3M, including but not limited to 0.3M, 0.4M, 0.5M,0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.5M, 1.75M, 2M, 2.5M, and 2.75M

In further embodiments, the buffer contains one or more metal ions addedat a concentration of about 10 mM or less, to about 100 mM or more,including but not limited to: 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM,80 mM, and 90 mM. In some embodiments, the metal ions are zinc and/orgold, including but not limited to ZnSO₄ and AuCl₄.

Methods of Using Reflectins and/or RRPs

Any methods of using proteins that reflect and/or have a high refractiveindex can be used. Some methods include, but are not limited to: as areporter protein to characterize transcription of a protein and/or apromoter, in nanostructured supramolecular devices, and fornanofabrication of any type of material, for example reflectivebiomaterials.

EXAMPLES

In Examples 1-3, a total of 6 different reflectin proteins from theSquid E. scolopes were identified and sequenced. Example 4 provides ananalysis of the sequence. In Examples 5-12, the smallest active portionof the protein, the Reflectin Repeat Peptide (RRP) is expressed,identified and characterized. In Examples 13-14 various methods for theuse of the peptides and proteins are provided.

The specimens of E. scolopes were obtained from the shallow reef flatsof Oahu, Hi., transported to circulating natural seawater aquaria at theUniversity of Hawaii, and maintained as described in Weis, et al. Biol.Bull. 1993, 184, p. 309 (herein incorporated by reference in itsentirety). All chemicals were obtained from Sigma-Aldrich (St. Louis,Mo.) unless otherwise noted.

Example 1 Isolation of Reflectin Proteins from E. scolopes

To enrich for the reflecting, the light organ reflector (LOR) was firsthomogenized in 50 mM sodium phosphate buffer, pH 7.4, with 0.1 M NaCl(PBS) in a ground glass homogenizer on ice to extract the aqueoussoluble fraction. The total homogenate was centrifuged at 20,800×g for15 min at 4° C. The resulting supernatant was removed. The pelletedmaterial was then washed by repeatedly resuspending it in PBS andcentrifuging the resuspension at 20,800×g for 15 min at 4° C. tore-pellet the aqueous-insoluble material. The resulting washed pelletwas resuspended in 2% SDS in PBS to extract the SDS-soluble fractionthat contained the reflecting. The suspension was then centrifuged, asdescribed above, and the supernatant retained for analyses.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wascarried out on a Bio-Rad Mini-Protean II electrophoresis system(Bio-Rad, Hercules, Calif.) under standard SDS-PAGE procedures (modifiedfrom Laemmli, Nature 227, p. 680). The protein concentrations of allfractions were determined spectrophotometrically.

Extractions of total protein from the E. scolopes LOR revealed a set ofthree abundant polypeptides that resolved between 33-36 kDa on SDS-PAGEand were a characteristic golden-yellow color upon silver staining (FIG.1E, E′). These polypeptides (reflecting) were not detected in theaqueous soluble fraction of the LOR, but were abundant in thesupernatant of the SDS-solubilized pellet, composing ˜40% of theproteinaceous component of the LOR.

To determine the approximate concentrations of reflectins in the LOR,the protein concentration of the whole homogenate, PBS-solublesupernatant, and the SDS-soluble fraction of the light organ reflectorwere each determined. From SDS-PAGE analysis of the SDS-soluble fractionof the LOR, reflectins made up at least about 50% of the total proteinin this fraction. This value was used to back-calculate to determinewhat proportion of the whole homogenate was reflecting.

Example 2 Tissue Localization of Reflectins

To localize the reflecting within the LOR, polyclonal antibodies weregenerated against gel-purified reflectin proteins (FIG. 1E, lane 5) andused in immunocytochemical and immunoblot analyses.

To generate material for antibody production, the reflectin proteinswere purified directly from the SDS-PAGE gel of the LOR proteins.Briefly, the 2% SDS-soluble fraction of light organ reflectors fromseven adult animals was applied to a stacking gel without wells. Theportion of the gel containing the reflectins was excised from the geland homogenized in 2% SDS in PBS. The resulting slurry was transferredto a Microcon-100 spin filter (Millipore Corp., Bedford, Mass.) andcentrifuged at 3000×g for 10 min at room temperature. An aliquot of thefiltrate was resolved on SDS-PAGE, that indicated that the desiredproteins had been isolated. The remaining filtrate protein was used inthe production of polyclonal antibodies (Covance Research Products,Inc., Denver, Pa.). For western blot analysis, protein extractions ofthe SDS-soluble pellet of various adult tissues were performed asdescribed above for the LOR. The protein concentration of eachextraction was determined spectrophotometrically and SDS-PAGE gels wererun according to standard protocol. The companion SDS-PAGE gel wassilver stained according to standard procedures. For western blots,proteins were electrophoretically transferred to nitrocellulose membrane(Bio-Rad, Hercules, Calif.). Blots were blocked overnight in 4% milk in50 mM Tris, 150 mM NaCl, 0.5% Tween 20, pH 7.5 (TTBS). Following thisblocking step, blots were incubated for 1 h in a 1:10,000 dilution ofantiserum in 1% milk/TTBS. Blots were washed 3 times in 1% milk/TTBS andthen incubated for 45 min in a mixture of 1:3000 goat anti-rabbitsecondary antibodies conjugated to horseradish peroxidase (Bio-Rad,Hercules, Calif.) and 1:3300 avidin-conjugated horseradish peroxidase todetect biotinylated molecular mass markers (Bio-Rad, Hercules, Calif.).These detection reagents were diluted in 1% milk/TTBS. Detection ofcross-reactive bands was achieved by chemiluminescence (ECLchemiluminescence kit, Amersham Biosciences Corp, Piscataway, N.J.).

Immunogold localization by transmission electron microscopy (TEM) wasperformed as follows: Light microscopy and TEM of squid tissues wereperformed as described in McFall-Ngai, et al. (Biol. Bull., 1990, 184,p. 296) and Montgomery et al. (J. Biol. Chem., 1992, 267, p. 20999),each of which is herein incorporated by reference in its entirety.Immunocytochemistry at the TEM level was also performed as described inWeis et al (see Example 1) except that anti-reflectin antibodies wereused as the primary antibody at a 1:1000 dilution. A 1:50 dilution ofgoat anti-rabbit IgG conjugated to 15-nm gold spheres (Ted Pella,Redding, Calif.) was used as the secondary antibody. To control fornonspecific binding of the secondary antibody, a subset of the grids wasincubated with a 1:1000 dilution of preimmune serum. TEM was performedon a JEOL 100 CX transmission electron microscope at the University ofSouthern California (Los Angeles, Calif.).

The reflectin antibodies strongly recognized the LOR, but not thebacteria-containing epithelium, the ink sac, or lens of the light organ(FIG. 2A). The results of the immunogold localization showed that thereflectins in the LOR were directly associated with the LOR platelets(FIG. 2A′) and that no labeling was detected in the surroundingconnective tissue or ink granules (FIG. 2A′). Higher magnification TEMimages of the LOR demonstrated that the antibodies cross reactedspecifically with the electron-dense platelets but not theinter-platelet region (FIG. 2A′, inset).

Proteins with similar molecular mass, biochemistry, and antigenicity toLOR reflectins were found in all reflective tissues of E. scolopes.Silver-stained SDS-PAGE gels revealed that the characteristicgolden-yellow bands at 33-36 kDa were detectable in reflective tissues.In addition, these proteins cross reacted with antibodies to LORreflectins in western blot analyses (FIGS. 1A, 2B). Cross-reactive bandswere also found at other molecular weights (26, 55, and 120 kDa), andcorresponding golden-yellow bands were present on the SDS-PAGE gels ofthese tissues. Because the antibodies were generated from a verydiscrete region of a gel, and these other bands only occur in reflectivetissues, it is likely that the antibodies were cross reacting with othermembers of the reflectin family or closely related proteins. Crossreactivity was low or undetectable in non-reflective tissues (gills,muscle, eye lens, and light organ lens) (FIG. 2B).

Example 3 Sequencing and Analysis of Reflectins Genes

The sequences of three tryptic peptides (FIG. 3A) from tryptic digestionof gel-purified reflectins (FIG. 1E, lane 5) were used to identifyreflectin cDNAs from predicted translations of E. scolopes cDNA and ESTlibrary clones as described below. The tryptic peptides had ambiguitiesin them as expressed by an “X” in the following sequences:SMFNYGWMMDGDR, (SEQ ID NO:31) EGYYPNYSYGR, (SEQ ID NO:32) andYFDMSNWQMDMQGR. (SEQ ID NO:33)

Protein extracts from light organs were subjected to SDS-PAGE. Reflectinbands were excised from the gel and subjected to trypsin digestion. Theresulting tryptic peptides were sequenced by mass spectrometry (HarvardMicrochemistry Facility, Cambridge, Mass.). The amino acid sequences ofthree tryptic peptides were used to screen predicted translations ofsequences from cDNA pools constructed from the light organs of juvenileanimals (SEQ ID NOs: 31-33). One partial sequence was obtained from thispool, the translation of which contained 2 of the 3 tryptic peptidesequences. This small tryptic peptide sequence had significantsimilarity (88%) to Loligo forbesi ‘methionine-rich repeat protein 1’(mrrp1; accession no. CAC86921) (SEQ ID NO:14).

Using the nucleotide sequence of L. forbesi mrrp1 (SEQ ID NO:13) forinformation about possible length and the sequence of the E. scolopespartial clone for primer design (Table 2), RACE-PCR (rapid amplificationof cDNA ends-polymerase chain reaction) was conducted on an E. scolopeslight organ cDNA pool to obtain full-length clones. 5′ and 3′ RACE-PCRswere performed on the clone using the SMART RACE cDNA amplification kit(BD Biosciences Clontech, Palo Alto, Calif.) and primers specific to thecDNA clone (see Table 2; 33F3, 33R2, 33R3, 33R4). First-strand synthesiswas performed on 195 ng of light organ mRNA according to themanufacturer's instructions. Both 5′ and 3′ RACE reaction conditionswere as follows: 5 cycles of 94° C. for 30 sec, 72° C. for 3 min; 5cycles of 94° C. for 30 sec, 70° C. for 30 sec, 72° C. for 3 min; 25cycles of 94° C. for 30 sec, 68° C. for 30 sec, 72° C. for 3 min. RACEproducts were run on 1% agarose gels and stained with ethidium bromideaccording to standard procedures. RACE products were gel-extracted(GeneClean kit, Bio101, Carlsbad, Calif.) and ligated into the pGEM-Teasy vector (Promega Corp., Madison, Wis.). Products from the ligationreactions were transformed into E. coli DH5alpha and transformants werescreened for inserts by blue-white screening on LB-carbenicillin (50μg/ml) plates containing 0.9 mg IPTG and 800 μg X-gal (Promega Corp,Madison, Wis.). White colonies were further screened by restrictionenzyme digestion (EcoRI) to identify those transformants with plasmidsthat contained appropriately sized inserts. Plasmids from positivecolonies were mini-prepped (Qiagen Inc., Valencia, Calif.) and sequencedat the University of Hawaii Biotechnology/Molecular BiologyInstrumentation and Training Facility. TABLE 2 Primers used for RACE-PCRand standard PCR reactions. SEQ ID Primer Primer type Direction Sequence(5′ to 3′) NO: 33F3 3′ RACE forward CGC CAC TGC AAC CCG TAT AGC CAA TGG34 33R2 5′ RACE reverse CCA ATA GGG GCT GCA GTA GCG TCC 35 33R3 5′ RACEreverse GTT GCC GGA GCG GTT CCA GTG GTT GTA A 36 33R4 5′ RACE reverseCCC GGG GTA GTT CCA GTA TCT GCC AT 37 33AF standard PCR forward ATG AACCGT TTT ATG AAC AGA TAC CG 38 33BF standard PCR reverse ATG AAC CGT TACATG AAC CGA TTC CG 39 33A1R standard PCR reverse GTA ATA GTC GTT CAT TCCGTA TTG GTC C 40 33B1R standard PCR reverse GAG CAA GAC GTT CAA GAA TTTCAG ACG 41 33B2R standard PCR reverse CCA GTT GTA ATA ATT ATA GGG ATAATC C 42 33BR standard PCR reverse CCA TGT ATC GTC CCT GCA TGT CCA TCC43

Genomic DNA extracted from the light organ of a single adult E. scolopeswas used as a template for PCR reactions to: i) determine whether genesfor all 6 cDNAs occur in the genome, or are the result of alternativesplicing or allelic differences; and, ii) provide information about genestructure. To amplify reflectin genes from genomic DNA, PCR reactionswere carried out using all possible combinations of 2 forward primers:33AF(SEQ ID NO:38), or 33BF(SEQ ID NO:39) and 4 reverse primers: 33A1 R(SEQ ID NO:40), 33B1R(SEQ ID NO:41), 33B2R(SEQ ID NO:42), and 33BR(SEQID NO:43); (see Table 2). Reactions were carried out with 1.5 mM MgCl₂,1 μM each forward and reverse primers, 1 mM dNTPs, and 2.5 U Taq DNApolymerase (Promega Corp., Madison, Wis.). Reaction conditions were asfollows: 94° C. for 2 min; 94° C. for 30 sec, 55° C. for 30 sec, 72° C.for 1.5 min for 35 cycles; 72° C. for 5 min. PCR products were clonedand sequenced as outlined above for RACE products.

In addition to reflectin gene sequences obtained from light organ cDNAand genomic DNA, sequences were obtained from an EST database beingconstructed from light organ cDNA libraries of juvenile animals.

All six full-length reflectin sequences contained stop codons followedby polyadenylated tails, that demonstrates that there was no genomic DNAcontamination. Accession numbers for the reflectins can be found inTable 3.

To produce the cDNA pool, RNA was isolated from the light organs fromjuvenile E. scolopes that were dissected, placed in RNAlater (Ambion,Inc., Austin, Tex.), and stored at −20° C. Total RNA was extracted asfollows: 80 juvenile light organs were homogenized in 600 μl TriPureIsolation Reagent (Roche Applied Sciences, Indianapolis, Ind.) for 30min on ice in a ground glass homogenizer. The homogenate was incubatedfor 5 min at room temperature, and then 120 μl of chloroform was addedto the homogenate. The mixture was allowed to stand for an additional 10min at room temperature, and then centrifuged at 12,000×g for 15 min at4° C. The upper aqueous phase was transferred to a new tube and 300 μlof isopropanol was added. This mixture was incubated for 7 min at roomtemperature to allow precipitation of total RNA and then centrifuged at10,800×g for 10 min at 4° C. The supernatant was discarded and the RNApellet was washed once with 75% ethanol. The pellet was air dried andwas then resuspended in 50 μl of RNase-free water. The resuspension wasthen incubated at 55° C. for 15 min and assessed for quantity and purityspectrophotometrically. mRNA was extracted from total RNA using the MPGmRNA Purification Kit (CPG, Lincoln Park, N.J.) according to themanufacturer's instructions. mRNA was quantified, assessed for purityspectrophotometrically, and resolved on a 1% agarose gel to confirm thatthe RNA was not degraded.

Genomic DNA was extracted from one adult light organ reflector using theportion of the MasterPure Complete DNA and RNA Purification Kit(Epicentre, Madison, Wis.) designed to isolated the DNA. During theextraction, the sample was treated with 5 μg RNase A to digestsingle-stranded RNAs. The DNA was quantified and assessed for purityspectrophotometrically. TABLE 3 Comparison of predicted proteincharacteristics of reflectins 1a-3a and L. forbesi mrrp1 (Lf). Accessionmolecular SEQ ID number # residues mass (kDa) pI NO 1a AY294649 283 36.78.84 2 1b AY294650 282 36.2 8.82 4 2a AY294652 283 36.7 8.84 6 2bAY294653 284 37.0 8.80 8 2c AY294654 284 37.2 8.81 10 3a AY294651 28837.6 8.81 12 Lf CAC86921 264 32.8 7.61 14# residues, number of total amino acids;pI, predicted isoelectric point.

Example 4 Analysis of Reflectin Sequences and Homologies

RACE-PCR conducted on light organ cDNA pools using reflectin primers(Table 2) identified six similar reflectin cDNAs (FIG. 1; Table 3),suggesting that several genes encoding reflecting were expressed in thelight organ. Sequencing of PCR products from the amplification ofgenomic DNA provided evidence that all six reflectin genes amplifiedfrom the cDNA pools are represented in the genome of a singleindividual. None of the reflectin genes amplified from genomic DNApossessed introns. Only one entry in the nucleotide databases hadsimilarity to the E. scolopes reflecting, a gene sequence from theEuropean squid Loligo forbesi (Weiss et al. NCBI accession numberCAC86921, 2002) that encodes ‘methionine-rich repeat protein 1’ (FIG. 1,A and B; Table 3) (accession no. CAC86921). However, there was no knownfunction for L. forbesi mrrp 1, and this protein was less than 72.5%identical to any of the E. scolopes sequences, while the reflecting fromE. scolopes were between 85 and 98% identical to each other (see FIG.4B).

The derived amino acid sequences of the six full-length clones werealigned, demonstrating that the reflecting are highly similar(85.0-98.6%) and group into three subfamilies (FIG. 4A and B; Table 3).Analysis of their structure revealed some unusual characteristics.Reflectins possess a highly unusual amino acid composition (FIG. 4C);six amino acids (Y, M, R, N, G, D) compose over 70% of the total, andfour other amino acids (A, I, L, K) are absent. Although their SDSsolubility suggested that they can be membrane associated, furtheranalyses demonstrated that they are not predicted to possess hydrophobicor charged clusters, transmembrane domains, orglycosylphosphatidylinositol anchors. However, each reflectin iscomposed of five repeating domains (FIG. 4). When these repeats arealigned (FIG. 3A, lower), a ‘core’ subdomain (SD) of 18-20 amino acidsis revealed; the subdomains were defined by the presence of a repeatingmotif [α(X)_(4/5)MD(X)₅MD(X)_(3/4)]. that occurs in 4 of the 5subdomains. In this motif, X represents any amino acid; the subscriptednumbers represent the number of amino acids at that position and theslash represents “or.” In these subdomains 21 of 23 methionine residuesoccur in the same relative position in the repeat. The subdomains areenriched in M, R, G, D, S, and Q, and depleted in Y, N, W, P, and Frelative to the whole protein (Table 4). Inter- and intra-proteinsubdomain alignments demonstrated that individual subdomains fromdifferent reflecting (e.g., SD1 from 1a vs. SD1 from 2a) are moresimilar to each other (80-100%) than are different subdomains (e.g., SD1from 1a vs. SD2 from 1a) of the same reflectin (55-70%) (FIG. 4B). Thesedata suggest greater functional constraint on the sequence within asubdomain across the family. TABLE 4 Amino acid composition of thesubdomains of reflectins and comparison to amino acid composition of thecomplete protein. Reflectin 1a was used as a representative protein.outside depleted (−) or amino in subdomains subdomains in total proteinenriched (+) in acid # % # % # % subdomains Y 9 9.2 50 27.0 59 19.8 − M23 23.5 19 10.3 42 14.6 + R 22 22.4 11 5.9 33 11.8 + N 4 4.1 24 13.0 289.7 − G 11 11.2 13 7.0 24 8.3 + D 17 17.3 6 3.2 23 8.3 + W 4 4.1 7 3.811 5.9 − S 7 7.1 8 4.3 15 4.9 + P 0 0.0 13 7.0 13 4.5 − Q 10 10.2 3 1.613 4.5 + F 2 2.0 7 3.8 9 2.8 − E 0 0.0 4 2.2 4 1.4 − H 0 0.0 4 2.2 4 1.4− C 0 0.0 4 2.2 4 1.4 − T 0 0.0 3 1.6 3 0.3 − V 0 0.0 1 0.5 1 0.3 − A 00.0 0 0 0 0.0 na I 0 0.0 0 0 0 0.0 na L 0 0.0 0 0 0 0.0 na K 0 0.0 0 0 00.0 nain subdomains, amino acid representation within all 5 subdomains (n = 98amino acids);outside subdomains, amino acid representation outside all 5 subdomains(n = 185 amino acids);in total protein, representation of each amino acid in the entiresequence of the protein (n = 283 amino acids);#, occurrence of each amino acid;%, percent occurrence of each amino acid;na, not applicable.

The derived amino acid sequences of the six full-length reflectin cloneswere aligned, and all three tryptic peptides from reflectin proteinsequencing were found in the translation of all clones (FIG. 4Aunderlined sequences). The alignment demonstrates that the proteins arevery similar to one another as well as to the L. forbesi mrrp1 (FIG.4A). Pairwise comparisons of all six sequences suggests a grouping ofthe E. scolopes reflectins into three subfamilies, reflectins 1, 2 and 3(FIG. 4A and B), based on percentage identity of the amino acids and thepositions of deletions/insertions.

As shown in FIG. 5, alignment of the polynucleotide sequences results inconsiderably less identity. For example, E. scolopes reflectins 1a, 2a,3a, and L. forbesi share only 63.5% identity. Identity between the E.scolopes proteins is better than with L. forbesi, resulting in about77.2% identity.

The E. scolopes reflectins have theoretical masses between 36.2 and 37.6kDa, predicted isoelectric points between 8.80-8.84 (Table 3), and ahighly unusual amino acid composition (FIG. 4C). Comparing thereflectins with other proteins by available protein analysis algorithmsrevealed that the reflectins possess extremely high usage of Y, M, andR, a high usage of W, and an extremely low usage of T, V, A, I, L, andK. The reflectins possess one of the highest tyrosine contents (19.8%)among current Protein Database proteins. Other tyrosine-rich proteinsinclude an assortment of extracellular matrix and structural proteinsincluding insect storage proteins (11-27%), keratin-associated proteins(12-17%), dental enamel peptides (13%), tyrosine-rich acidic matrixproteins (10%), and glycoproteins involved in oocyst cell wall formationin apicomplexan parasites (10%), suggesting that this amino acid may beimportant in the formation of protein superstructures.

Application of algorithms that predict secondary structure revealed highrepresentation of order-promoting residues (W, Y, F, and N), the absenceof 4 residues (A, I, L, and K) that are abundant in other proteins andoften used in packing of hydrophobic cores, and the presence of highlyordered repeats suggest that the reflectins fold and pack in unusualways.

Analysis of reflectin clones was carried out using MacVector 7.1.1(Accelrys, San Diego, Calif.). Sequence alignments were performed byClustalV. Amino acid sequence analysis was performed using the followingprograms available on the ExPASy Molecular Biology server(www.ExPASy.org): SignalP, PredictProtein, TMHMM, ProtParam, Radar,SAPS, nnPredict, Jpred, Sulfinator, and big-PI Predictor.

The identification and characterization of the reflectins confirmed thatwhile the majority of animal reflective tissues are composed of purineplatelets, cephalopod reflector platelets are proteinaceous. Reflectins,a previously undescribed protein family with skewed amino acidcompositions, repeating domains, and localized deposition, are thus farrestricted to cephalopods. They represent a striking example of naturalnanofabrication of photonic structures in these animals.

Interestingly, a seventh reflectin protein sequence was identified andcalled reflectin 2d (SEQ ID NOs: 45 and 46) because of its homology tothe other reflectin 2 proteins. Reflectin 2d was amplified from genomicDNA but has not yet been identified in the light organ cDNA pool. On anamino acid level, it is 89.7% identical to 1a, 86.4% identical to 1b,98.9% identical to 2c, 96.0% identical to 3a and 70.5% identical to L.forbesi mrrp. This reflectin appears to have resulted from genomicamplification and has not yet been shown to be expressed in the LOR.

To further analyze the polypeptides and to determine the minimumfunctional or active portion, an RRP was prepared as described below andanalyzed.

Example 5 Structural Characterization of the Reflective Repeat PeptideFrom the Reflectin 1A Gene

The 18 amino acid synthetic RRP (New England Peptide) shown in FIG. 6(SEQ ID NO:15) was resuspended in water to a final concentration of 10mg/ml. Secondary structure determination was undertaken using acombination of Circular Dichroism (CD) and X-ray Diffraction (FIG. 7).The CD spectrum of the suspension revealed a mostly beta-sheet characterwith peak absorbance at 207 nm in the far-UV. The CD spectrum of proteinin the near-UV (250-350 nm) can be sensitive to certain aspects oftertiary structure. At these wavelengths the chromophores are thearomatic amino acids and disulfide bonds, and the CD signals theyproduce can represent a defined tertiary structure of the protein.Signals in this region are attributed to tyrosine (270-290 mn),tryptophan (280-300 nm), and disulfide bonds that give rise to a broadbut weak signal throughout the near-UV spectrum. The presence of astrong near-UV signal in the CD spectrum for the RRP was an indicationthat the protein was folded into a well-defined orientation with adefined tertiary structure in solution. To complement the CD data, theresuspended solution was dried in a glass capillary for X-raydiffraction analysis. After drying, the protein remained optically clearand gave rise to distinct rings in the X-ray diffraction pattern at0.38, 0.46, and 1.15 nm, indicative of a crystalline beta-sheetstructure. The fact that the peptide maintained its overall secondarystructure following the drying process was due to the stability of thepeptide interactions.

Example 6 LVTEM Analysis

In order to determine the contribution of the peptide in higher-orderedtertiary and quaternary structures, electron microscope studies of thepeptide spotted onto copper grids with an amorphous carbon support wereperformed. A combination of Low-voltage transmission electron microscopy(LVTEM) and High-voltage transmission electron microscopy (HVTEM) wasused to characterize the peptide. Low-voltage electron microscopyallowed for imaging the protein materials without staining and preventedbeam damage to otherwise delicate structures. LVTEM micrographs of newlyresuspended RRP and peptide that was allowed to sit for several weeksafter resuspension revealed a dramatic difference in the overallcrystallinity and structure of the material. FIG. 8 shows the LVTEMimages of the two samples. The newly resuspended RRP showed distinctstrands and small spheres. The imaged strands were ˜8 nm in diameter andthe small spheres measure ˜10 nm in diameter. LVTEM of the aged samplerevealed a dramatically more electron dense sample and only sphericalstructures with diameters of ˜12 nm were resolved from the image. Thestructure of the more electron dense regions was not resolved due to thenature of the lower electron voltage used by the microscope.Corroboration for the filamentous nature of newly resuspended RRP wasobtained using Atomic Force Microscopy (AFM). The RRP was spotted onto asilicon wafer and allowed to dry in air. The AFM image showed longaligned fibrils that extended for several microns (FIG. 8). A FastFourier-Transformed (FFT) image of the AFM topographic image revealedthat the spacing between crystals was regular and equal to 8 nm onaverage, revealing periodicity of the native RRP protein.

Example 7 Solubility Of Reflectins

To determine the relative solubility of reflectins, insoluble proteinswere extracted from Euprymna scolopes light organ reflector (LOR) or eyereflectors by homogenization in 50 mM sodium phosphate buffer, pH 7.4,with 0.1 M NaCl (PBS) on ice. The total homogenate was centrifuged at20,800×g for 15 min at 4° C. The pelleted insoluble material was thenwashed repeatedly in PBS. After the final wash, the material wasresuspended in PBS. The resuspension was aliquotted to individual tubes.Each tube was centrifuged at 20,800×g for 15 min at 4° C. to re-pelletthe insoluble material. The supernatant was discarded. The resultingpellet was resuspended in 25 μl of treatment solution and centrifugedagain. The supernatant was removed, mixed with SDS-PAGE buffer, boiledfor 5 min, and subjected to SDS-PAGE. Relative solubility in thepresence of various reagents (Table 5) was assessed by comparison with acontrol sample that had been solubilized in 2% SDS. TABLE 5 Solubilityof reflectins in various reagents. relative solubility Treatment ofreflectins 0.1% SDS − 0.2% SDS ++ 1-2% SDS +++++ 1% Triton X-100 − 0.5%Tween-20 − 1% CHAPS − 1M nondetergent sulfobetaine (NDSB)-195 + 1MNDSB-195 + 2% CHAPS + 1M NDSB-201 ++ 1M NDSB-201 + 2% CHAPS + 1MNDSB-256 + 1M NDSB-256 + 2% CHAPS − 1 M urea − 3 M urea − 3 M urea, 0.3M NaCl + 8 M urea − 8 M urea, 2% CHAPS ++ 50% dimethylsulfoxide − 100%dimethylsulfoxide ++++ 100% trifluoroethanol ++ 100% acetonitrile − 0.5M EDTA + 1 M ammonium acetate ++ 1 M ammonium sulfate ++ 1 M lithiumchloride + 5 M lithium chloride + 2 M sodium chloride in PBS, pH 7.4 + 3M sodium chloride in PBS, pH 7.4 + 0.25 M sodium sulfate, pH 6 − 0.25 Mammonium carbonate, pH 8.5 − 0.1 M sodium carbonate, pH 11 +

This information can be used in combination with the information gainedin other Examples herein to identify the best conditions for the use,native conformation, reflectivity and solubilization of the RRPproteins.

Example 8 HVTEM Analysis

A more detailed inspection into the molecular structure was undertakenusing HVTEM. Newly resuspended RRP was largely unstable in thehigh-voltage beam and a high-resolution image was not obtainable. Theaged peptide, with higher electron density in the LVTEM beam, wassignificantly more stable in the higher voltage beam and an underlyinghigh-resolution structure was obtained. FIG. 9 shows thesehigh-resolution images at two different magnifications. Within the RRPstructure, there existed both the spherical and fibril structures notedin the LVTEM study, albeit with a tighter association betweensubstructures. The interaction between these two structures lead to alarge thread-like formation that could span between 30 and 200 nm. Dueto the increased beam stability, a distinct and repeatable electrondiffraction pattern was resolved and is included in FIG. 9.

Incorporation of inorganic metals to the native reflectin basedplatelets was theorized to provide structural stability and/or anincrease in overall effective refractive index of the material. This wasexplored in Example 9.

Example 9 Interaction with Metals

Ultra-thin sections of the reflectin platelets are obtained. HRTEM andelemental analysis of these tissues reveals whether inorganic metals area necessary inclusion for protein stability and contribute to theeffective refractive index of the material.

Due to the unique amino acid composition of the RRP, certain inorganicmetals can be used to affect their structure and effective refractiveindex. 50 mM ZnSO₄ was added to the RRP and spotted on a copper grid forLVTEM analysis. Zn addition altered the overall structure of the RRP asseen in FIG. 10. The fibrils following Zn addition had a slightly widerdiameter of ˜15 mn and seemed to possess a greater rigidity. Zn is knownto be important in control of both tertiary and quaternary structure ofproteins and this result suggests that it plays a role in the structureof the RRP. Tyrosine residues, which are found in a high percentage ofboth the RRP and the full length reflectin proteins, have beendemonstrated previously to potentiate the reduction of AuCl₄ to metallicgold with a high degree of crystallinity. Following AuCl₄ addition toRRP, there was an overnight precipitation of the RRP in solution. LVTEMand HVTEM images of the RRP-Au precipitate were analyzed. HRTEM imagesshowed that the gold was able to incorporate within the microstructuralframework of the RRP. Spherical gold nanoparticles were embedded withinthe proteinaceous material and had similar dimensions (˜12 nm) of thespherical structures observed for aged RRP (FIG. 9). Composition of thenanoparticles were confirmed to be crystalline Au⁰ by indexing therefraction pattern from these regions and measuring the lattice spacingof the nanoparticles from the high resolution image. In addition,composition of the electron-dense threads was confirmed to be acomposite inorganic-organic material of RRP and Au from energydispersive spectroscopy. The ability to incorporate inorganic metals byusing the RRP as a template showed that this approach can be used tocontrol index of refraction of the composite material and tune thematerial appropriately.

Bragg reflection from native platelets is hypothesized to beaccomplished through the use of a high index of refraction proteinmaterial. The unique amino acid contribution of the reflectin proteinsto include the aromatic residues tyrosine and tryptophan is likely tocontribute to the overall bulk refractive index. The use of inorganicsto increase the bulk refractive index was also discussed above. Whilethe bulk characteristics are integral in the reflection process, it islikely that there exists a high degree of crystallinity of the proteinand/or protein-inorganic matrix and is necessary in the reflectionmechanism. Protein concentrations within the reflective organelles wouldhave to be extremely high to produce the necessary refractive indexmismatch with the outlying cellular components. Furthermore, if theprotein bulk was generally amorphous, there would exist a high level ofscatter within the reflectin organelle and reduce the overallreflectivity of the platelets. To circumvent this problem, theproduction of protein matrix with a high degree of crystallinity wouldgenerate a material of extremely high protein concentration with littlescatter and high reflection. To determine conditions necessary forcrystallization of the RRP, a number of different conditions wereexplored using a hanging-drop vapor diffusion protein crystallizationtechnique. This combinatorial approach showed that in almost 20% of theconditions, the RRP precipitated out of solution within a day. Withinthe precipitate, the RRP appeared to possess clear regions that appearedreflective under an optical microscope with overhead illumination asseen in FIG. 11. These samples were transferred to a glass slide andviewed under a cross-polarizing microscope to determine birefringence ofthe material. Indeed, the material demonstrated significantbirefringence as associated with ordered materials. Reversiblecrystallization can lead to the development of a tunable Braggreflecting system by controlling the levels of scattered and reflectedlight.

The characterizations from Examples 6-9 show that the RRP sequence hasthe potential for mineralization/metallization of inorganics and thatcertain inorganics can be involved in folding (tertiary structure) ofthe peptide and/or the effective refractive index.

Example 10 Production of a Bacterial Expression System for Reflectin 1A

In order to explore the full-length protein complex and produce largequantities of the protein, a reflectin protein that was based on theamino acid sequence from reflectin la was recombinantly expressed (SEQID NOs: 1 and 2). A synthetic gene based on this sequence was producedbecause of the difficulty associated with polymerase chain reactionamplification of proteins with repetitive sequences and the largearginine content of the sequence. Because of the codon bias for E. coli,DNA sequences possessing rare codons not used by this organism are noteffectively recombinantly expressed. The sequence was optimized so thatit best reflected the codons used natively by E. coli. Initialrecombinant experiments have shown that the reflectin 1a sequence can beexpressed using an IPTG induced BL21 system. SEQ ID NOS: 44 and 45provide the nucleotide sequence of these constructs. FIG. 12 shows theexpression of this sequence and one that contains a N-terminalhexahistidine fusion to aid in subsequent purification (SEQ ID NO:45).Although in many buffers the protein is predominantly insoluble, theanalyses disclosed herein permit selection of buffer components that canfacilitate refolding of the recombinant protein into its nativeconformation.

Example 11 Use of Reflectins for Nanofabrication

Protein-based nanofabrication is a frontier area in biomimetics, inwhich protein structures are engineered to be used asbiomaterials(Zhang, et al. Curr. Opin. Chem. Biol. 2002, Vol. 6, page865, herein incorporated by reference in its entirety). For example,numerous biomaterials can be either genetically altered to producereflectin and/or RRP proteins, including but not limited to wood, silk,cotton, flax and burlap. Alternatively, the purified reflectin and/orRRP protein can be admixed with a synthetic material to produce asemi-biomaterial, including but not limited to: polyester, metals,plastics, and the like.

Example 12 Use of Reflectins for Nanostructured Supramolecular Devices

Future embodiments of the invention provide reflectins that can supportthe ‘bottom-up’ synthesis of nanostructured supramolecular devices,especially those used in spectroscopic and optic applications (Vukusic,et al. Nature 2003, vol. 424, page 852, herein incorporated by referencein its entirety). For example, reflectin-based nanoreflectors can becoupled with artificial photosynthetic membranes (Steinberg-Yfach, etal., Nature 1997, Vol. 392, page 479, herein incorporated by referencein its entirety) or with bacteriorhodopsin-based bioelectronic devices(Wise, et al. Trends Biotechnol. 2002, Vol. 20, page 387, hereinincorporated by reference in its entirety) to enhance the power andefficiency of these systems.

Example 13 Use as a Reporter Gene

Currently, two of the most common reporter genes for use intranscriptional studies are green fluorescent protein (GFP) and βgalactosidase. Because of their unique qualities, any of the reflectinproteins or RRPs can be used in addition to these reporter genes or asan alternative. The RRP proteins are operably linked to any promoterknown to one of skill in the art that is being studied. The expressionof the promoter is analyzed with respect to the amount of RRP producedas measured by spectroscopy, UV absorption, or microscopy in thepresence of various filters.

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described can be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein.

Furthermore, the skilled artisan will recognize the interchangeabilityof various features from different embodiments. Similarly, the variousfeatures and steps discussed above, as well as other known equivalentsfor each such feature or step, can be employed in various combinationsby one of ordinary skill in this art to perform methods in accordancewith principles described herein.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the invention extends beyond the specifically disclosedembodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof. Accordingly, the invention is notintended to be limited by the specific disclosures of preferredembodiments herein, but instead by reference to claims attached hereto.

1. An isolated reflectin polypeptide comprising a sequence having atleast about 75% identity to SEQ ID NO:2.
 2. The isolated reflectinpolypeptide of claim 1, wherein said polypeptide has at least about 85%identity to SEQ ID NO:2.
 3. The isolated reflectin polypeptide of claim1, wherein said polypeptide has a predicted isoelectric point above 8.0.4. An isolated polynucleotide encoding a reflectin polypeptide, saidpolynucleotide comprising a sequence having at least about 65% identityto SEQ ID NO:1.
 5. An isolated polypeptide having and least one and nomore than four repeats of an amino acid sequence having the motif[α(X)_(4/5)MD(X)₅MD(X)_(3/4)], wherein α is MD, FD, or null; Xrepresents any amino acid; the subscripted numbers represent the numberof amino acids at that position; and the slash represents “or.”
 6. Theisolated polypeptide of claim 5, wherein said amino acid sequence isselected from the group consisting of: SEQ ID NOs: 15-30 and anycombination thereof.
 7. The isolated polypeptide of claim 5, whereinsaid polypeptide has an activity of a reflectin protein.
 8. An isolatedpolypeptide comprising six or more repeats of an amino acid sequencehaving the motif [α(X)_(4/5)MD(X)₅MD(X)_(3/4)], wherein α is MD, FD, ornull; X represents any amino acid; the subscripted numbers represent thenumber of amino acids at that position; and the slash represents “or.”9. The isolated polypeptide of claim 8, wherein said amino acid sequenceis selected from the group consisting of: SEQ ID NOs:15-30, and anycombination thereof.
 10. A biomimetic reflective material comprising afirst component, the first component comprising at least one polypeptideselected from the group consisting of: (a) a reflectin polypeptide; (b)a polypeptide having at least one and not more than four repeat units ofa reflectin polypeptide; (c) a polypeptide comprising at least sixrepeat units of a reflectin polypeptide; (d) an active or functionalhomologue or recombinant form of any of (a) through (c); and (e) anycombination of (a) through (d); the first component being in combinationwith at least a second component compatible with the first component,such that the combination forms a biomimetic reflective material. 11.The biomimetic reflective material of claim 10, the material comprisinga metal ion.
 12. The biomimetic reflective material of claim 10, thematerial having at least a first and a second refractive state, whereinthe material in the first refractive state has a refractive index thatis different from a refractive index of the material in the secondstate.
 13. A method of producing a biomimetic reflective material,comprising providing a first component comprising at least onepolypeptide selected from the group consisting of: (a) a reflectinpolypeptide; (b) a polypeptide having at least one and not more thanfour repeat units of a reflectin polypeptide; (c) a polypeptidecomprising at least six repeat units of a reflectin polypeptide; (d) anactive or functional homologue or recombinant form of any of (a) through(c); and (e) any combination of (a) through (d) combining the firstcomponent with at least a second component to form a biomimeticreflective material.
 14. The method of claim 13, wherein the secondcomponent comprises a member of the group consisting of: a metal, anion, a polymer, a fabric, a crystal, a fiber and a plastic.
 15. A methodof producing a biomimetic reflective material, comprising causingexpression in a cell, of at least one polypeptide selected from thegroup consisting of: (a) a reflectin polypeptide; (b) a polypeptidehaving at least one and not more than four repeat units of a reflectinpolypeptide; (c) a polypeptide comprising at least six repeat units of areflectin polypeptide; (d) an active or functional homologue orrecombinant form of any of (a) through (c); and (e) any combination of(a) through (d) using the cell or a fragment or extract thereof inproducing a biomimetic reflective material.
 16. The method of claim 15,wherein the cell is selected from the group consisting of: a plant cell,a bacterial cell; a fungal cell, and an animal cell.
 17. A method ofmodifying a refractive index of a reflectin, comprising: providing areflectin polypeptide in a composition compatible with a metal ion,wherein the composition has a first refractive index in absence of themetal ion; adding the metal ion to the composition, wherein thecomposition has a second refractive index in presence of the metal.